scholarly journals The H2O2 Stress-Responsive Regulator PerR Positively Regulates srfA Expression in Bacillus subtilis

2005 ◽  
Vol 187 (19) ◽  
pp. 6659-6667 ◽  
Author(s):  
Kentaro Hayashi ◽  
Taku Ohsawa ◽  
Kazuo Kobayashi ◽  
Naotake Ogasawara ◽  
Mitsuo Ogura
Keyword(s):  
Bacillus Subtilis ◽  
Promoter Region ◽  
Feeding Assay ◽  
Mobility Shift ◽  
Genetic Competence ◽  
Upstream Promoter ◽  
Upstream Promoter Region ◽  
Fusion Analysis ◽  
Gel Mobility Shift

ABSTRACT srfA is an operon required for the synthesis of surfactin and the development of genetic competence in Bacillus subtilis. We observed that the expression of srfA is downregulated upon treatment with H2O2. Thus, we examined the involvement of several oxidative stress-responsive transcription factors in srfA expression. Our DNA microarray analysis revealed that the H2O2 stress-responsive regulator PerR is required for srfA expression. This was confirmed by lacZ fusion analysis. A ComX feeding assay and epistatic analyses revealed that the role of PerR in srfA expression is independent of other known regulators of srfA expression, namely, comQXP, rapC, and spx. Gel mobility shift and footprint assays revealed that PerR binds directly to two tandemly arranged noncanonical PerR boxes located in the upstream promoter region of srfA. A transcriptional srfA-lacZ fusion lacking both PerR boxes showed diminished and PerR-independent expression, indicating that the PerR boxes we identified function as positive cis elements for srfA transcription.

Identification of functional TTF-1 and Sp1/Sp3 sites in the upstream promoter region of rabbit SP-B gene

2000 ◽  
Vol 278 (3) ◽  
pp. L477-L484 ◽  
Author(s):  
Ramgopal Margana ◽  
Kiflu Berhane ◽  
M. Nurul Alam ◽  
Vijayakumar Boggaram
Keyword(s):  
Promoter Region ◽  
Promoter Activity ◽  
Mutational Analysis ◽  
Surfactant Protein ◽  
Alveolar Type ◽  
Deletion Mapping ◽  
Clara Cells ◽  
Mobility Shift ◽  
Upstream Promoter ◽  
Upstream Promoter Region

Surfactant protein B (SP-B) is essential for the maintenance of biophysical properties and physiological function of pulmonary surfactant. SP-B mRNA is expressed in a cell type-restricted manner in alveolar type II and bronchiolar (Clara) epithelial cells of the lung and is developmentally induced. In NCI-H441 cells, a lung cell line with characteristics of Clara cells, a minimal promoter region comprising −236 to +39 nucleotides supports high-level expression of chloramphenicol acetyltransferase reporter activity. In the present investigation, we characterized the upstream promoter region, −236 to −140 nucleotides, that is essential for promoter activity. Deletion mapping identified two segments, −236 to −170 and −170 to −140 nucleotides, that are important for promoter activity. Mutational analysis and gel mobility shift experiments identified thyroid transcription factor-1, Sp1, and Sp3 as important trans-acting factors that bind to sequences in the upstream promoter region. Our data suggest that SP-B promoter activity is dependent on interactions between factors bound to upstream and downstream regions of the promoter.

scholarly journals ScoC Regulates Bacilysin Production at the Transcription Level in Bacillus subtilis

2009 ◽  
Vol 191 (23) ◽  
pp. 7367-7371 ◽  
Author(s):  
Takashi Inaoka ◽  
Guojun Wang ◽  
Kozo Ochi
Keyword(s):  
Bacillus Subtilis ◽  
Genetic Engineering ◽  
Genome Sequencing ◽  
Promoter Region ◽  
Sequencing Analysis ◽  
Transcription Level ◽  
Mobility Shift ◽  
State Regulator ◽  
Transition State Regulator ◽  
Gel Mobility Shift

ABSTRACT Bacillus subtilis mutants with high expression of the bacilysin operon ywfBCDEFG were isolated. Comparative genome sequencing analysis revealed that all of these mutants have a mutation in the scoC gene. The disruption of scoC by genetic engineering also resulted in increased expression of ywfBCDEFG. Primer extension and gel mobility shift analyses showed that the ScoC protein binds directly to the promoter region of ywfBCDEFG. Our results indicate that the transition state regulator ScoC, together with CodY and AbrB, negatively regulates bacilysin production in B. subtilis.

scholarly journals Regulation of the Bacillus subtilis ytmI Operon, Involved in Sulfur Metabolism

2005 ◽  
Vol 187 (17) ◽  
pp. 6019-6030 ◽  
Author(s):  
Pierre Burguière ◽  
Juliette Fert ◽  
Isabelle Guillouard ◽  
Sandrine Auger ◽  
Antoine Danchin ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Promoter Region ◽  
Sulfur Metabolism ◽  
Point Mutations ◽  
Constitutive Expression ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Mobility Shift ◽  
Common Motif ◽  
Gel Mobility Shift

ABSTRACT The YtlI regulator of Bacillus subtilis activates the transcription of the ytmI operon encoding an l-cystine ABC transporter, a riboflavin kinase, and proteins of unknown function. The expression of the ytlI gene and the ytmI operon was high with methionine and reduced with sulfate. Using deletions and site-directed mutagenesis, a cis-acting DNA sequence important for YtlI-dependent regulation was identified upstream from the −35 box of ytmI. Gel mobility shift assays confirmed that YtlI specifically interacted with this sequence. The replacement of the sulfur-regulated ytlI promoter by the xylA promoter led to constitutive expression of a ytmI′-lacZ fusion in a ytlI mutant, suggesting that the repression of ytmI expression by sulfate was mainly at the level of YtlI synthesis. We further showed that the YrzC regulator negatively controlled ytlI expression while this repressor also acted on ytmI expression via YtlI. The cascade of regulation observed in B. subtilis is conserved in Listeria spp. Both a YtlI-like regulator and a ytmI-type operon are present in Listeria spp. Indeed, the Lmo2352 protein from Listeria monocytogenes was able to replace YtlI for the activation of ytmI expression and a lmo2352′-lacZ fusion was repressed in the presence of sulfate via YrzC in B. subtilis. A common motif, AT(A/T)ATTCCTAT, was found in the promoter region of the ytlI and lmo2352 genes. Deletion of part of this motif or the introduction of point mutations in this sequence confirmed its involvement in ytlI regulation.

scholarly journals Cross-Regulation of the Bacillus subtilis glnRA and tnrA Genes Provides Evidence for DNA Binding Site Discrimination by GlnR and TnrA

2006 ◽  
Vol 188 (7) ◽  
pp. 2578-2585 ◽  
Author(s):  
Jill M. Zalieckas ◽  
Lewis V. Wray ◽  
Susan H. Fisher
Keyword(s):  
Bacillus Subtilis ◽  
Dna Binding ◽  
Promoter Region ◽  
Nitrogen Availability ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Amino Acid Sequences ◽  
Mobility Shift ◽  
Binding Domains ◽  
Gel Mobility Shift

ABSTRACT Two Bacillus subtilis transcriptional factors, TnrA and GlnR, regulate gene expression in response to changes in nitrogen availability. These two proteins have similar amino acid sequences in their DNA binding domains and bind to DNA sites (GlnR/TnrA sites) that have the same consensus sequence. Expression of the tnrA gene was found to be activated by TnrA and repressed by GlnR. Mutational analysis demonstrated that a GlnR/TnrA site which lies immediately upstream of the −35 region of the tnrA promoter is required for regulation of tnrA expression by both GlnR and TnrA. Expression of the glnRA operon, which contains two GlnR/TnrA binding sites (glnRAo1 and glnRAo2) in its promoter region, is repressed by both GlnR and TnrA. The glnRAo2 site, which overlaps the −35 region of the glnRA promoter, was shown to be required for regulation by both GlnR and TnrA, while the glnRAo1 site which lies upstream of the −35 promoter region is only involved in GlnR-mediated regulation. Examination of TnrA binding to tnrA and glnRA promoter DNA in gel mobility shift experiments showed that TnrA bound with an equilibrium dissociation binding constant of 55 nM to the GlnR/TnrA site in the tnrA promoter region, while the affinities of TnrA for the two GlnR/TnrA sites in the glnRA promoter region were greater than 3 μM. These results demonstrate that GlnR and TnrA cross-regulate each other's expression and that there are differences in their DNA-binding specificities.

The clpB gene of Bifidobacterium breve UCC 2003: transcriptional analysis and first insights into stress induction

Microbiology ◽  
2005 ◽  
Vol 151 (9) ◽  
pp. 2861-2872 ◽  
Author(s):  
Marco Ventura ◽  
John G. Kenny ◽  
Ziding Zhang ◽  
Gerald F. Fitzgerald ◽  
Douwe van Sinderen
Keyword(s):  
3D Structure ◽  
Binding Mode ◽  
Dna Interaction ◽  
Transcriptional Analysis ◽  
Amino Acid Residues ◽  
Mobility Shift ◽  
Bifidobacterium Breve ◽  
Gel Mobility Shift ◽  
Slot Blot Analysis

The so-called clp genes, which encode components of the Clp proteolytic complex, are widespread among bacteria. The Bifidobacterium breve UCC 2003 genome contains a clpB gene with significant homology to predicted clpB genes from other members of the Actinobacteridae group. The heat- and osmotic-inducibility of the B. breve UCC 2003 clpB homologue was verified by slot-blot analysis, while Northern blot and primer extension analyses showed that the clpB gene is transcribed as a monocistronic unit with a single promoter. The role of a hspR homologue, known to control the regulation of clpB and dnaK gene expression in other high G+C content bacteria was investigated by gel mobility shift assays. Moreover the predicted 3D structure of HspR provides further insight into the binding mode of this protein to the clpB promoter region, and highlights the key amino acid residues believed to be involved in the protein–DNA interaction.

Independent 5' and 3'-end determination of multiple dihydrofolate reductase transcripts

1987 ◽  
Vol 7 (10) ◽  
pp. 3732-3739
Author(s):  
J Y Yen ◽  
R E Kellems
Keyword(s):  
Dihydrofolate Reductase ◽  
Relative Abundance ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Overlapping Genes ◽  
Cytoplasmic Rna ◽  
Upstream Promoter ◽  
Upstream Promoter Region ◽  
Polyadenylation Sites

Multiple dihydrofolate reductase (dhfr) mRNAs, differing substantially in abundance, are produced as a result of the utilization of multiple transcription initiation sites and multiple polyadenylation sites. We have shown that dhfr mRNAs initiating from an upstream promoter region utilize the same collection of six polyadenylation sites and generate multiple dhfr mRNAs at the same relative abundance as do the mRNAs initiating from the major transcription promoter region. These results indicate that the 5' and 3' ends of dhfr mRNAs are independently determined. We show that the relative abundance of steady-state dhfr mRNAs was the same in nuclear and cytoplasmic RNA fractions. This finding makes it unlikely that differences in mRNA stability account for differences in the relative abundance of the multiple dhfr mRNAs in the cytoplasm. Our analysis of the dhfr promoter region revealed the existence of stable cytoplasmic polyadenylated transcripts complementary to the first 300 nucleotides of the dhfr transcripts initiating from the upstream promoter region. Therefore, the dhfr locus hosts two divergent and partially overlapping genes which share the same promoter region.

scholarly journals In Bacillus subtilis LutR is part of the global complex regulatory network governing the adaptation to the transition from exponential growth to stationary phase

Microbiology ◽  
2014 ◽  
Vol 160 (2) ◽  
pp. 243-260 ◽  
Author(s):  
Öykü İrigül-Sönmez ◽  
Türkan E. Köroğlu ◽  
Büşra Öztürk ◽  
Ákos T. Kovács ◽  
Oscar P. Kuipers ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Exponential Growth ◽  
Dna Microarrays ◽  
Antibiotic Production ◽  
Transcriptional Profiling ◽  
Mobility Shift ◽  
Carbohydrate Utilization ◽  
Altered Expression ◽  
Gel Mobility Shift

The lutR gene, encoding a product resembling a GntR-family transcriptional regulator, has previously been identified as a gene required for the production of the dipeptide antibiotic bacilysin in Bacillus subtilis. To understand the broader regulatory roles of LutR in B. subtilis, we studied the genome-wide effects of a lutR null mutation by combining transcriptional profiling studies using DNA microarrays, reverse transcription quantitative PCR, lacZ fusion analyses and gel mobility shift assays. We report that 65 transcriptional units corresponding to 23 mono-cistronic units and 42 operons show altered expression levels in lutR mutant cells, as compared with lutR + wild-type cells in early stationary phase. Among these, 11 single genes and 25 operons are likely to be under direct control of LutR. The products of these genes are involved in a variety of physiological processes associated with the onset of stationary phase in B. subtilis, including degradative enzyme production, antibiotic production and resistance, carbohydrate utilization and transport, nitrogen metabolism, phosphate uptake, fatty acid and phospholipid biosynthesis, protein synthesis and translocation, cell-wall metabolism, energy production, transfer of mobile genetic elements, induction of phage-related genes, sporulation, delay of sporulation and cannibalism, and biofilm formation. Furthermore, an electrophoretic mobility shift assay performed in the presence of both SinR and LutR revealed a close overlap between the LutR and SinR targets. Our data also revealed a significant overlap with the AbrB regulon. Together, these findings reveal that LutR is part of the global complex, interconnected regulatory systems governing adaptation of bacteria to the transition from exponential growth to stationary phase.

Identification of a cell-type-specific transcriptional repressor in the promoter region of the mouse hepatocyte growth factor gene

1994 ◽  
Vol 14 (11) ◽  
pp. 7046-7058
Author(s):  
Y Liu ◽  
A B Beedle ◽  
L Lin ◽  
A W Bell ◽  
R Zarnegar
Keyword(s):  
Epithelial Cells ◽  
Promoter Region ◽  
Nuclear Protein ◽  
Transcriptional Repressor ◽  
Cell Type ◽  
Mobility Shift ◽  
A Cell ◽  
Cell Type Specific ◽  
Gel Mobility Shift ◽  
Hepatocyte Growth

Hepatocyte growth factor (HGF), a cytokine with multiple functions, exhibits cell-type-specific as well as cytokine- and steroid hormone-regulated expression. The HGF gene is known to be expressed predominately in mesenchymal but not in epithelial cells. In this study, we report the identification of a cell-type-specific transcriptional repressor in the promoter region of the mouse HGF gene, which is evidently responsible for the suppression of HGF expression in epithelial cells. Gel mobility shift assays and DNase I footprinting studies revealed that a 27-bp element (-16 to +11) around the transcription initiation site is responsible for the binding of a nuclear protein which is present in epithelial but not in mesenchymally derived cells. Further analysis of the binding activity of the DNA region with nuclear protein revealed that an approximately 19-bp sequence containing a unique palindromic structure (5'-AACCGACCGGTT-3') overlapped by a CAP box is essential for binding. Substitution of a single base (the contact site) within this region by site-directed mutagenesis resulted in total abrogation of the binding of the nuclear protein and a concomitant increase in the transcriptional activity of various lengths of HGF-chloramphenicol acetyltransferase fused genes when transfected into the epithelial cell line RL95-2 but not the mesenchymal cell line NIH 3T3. Southwestern (DNA-protein) analyses revealed that the nuclear protein which binds to this repressor element is a single polypeptide of approximately 70 kDa. Analysis of the nuclear extract prepared from regenerating mouse liver at various times after two-thirds partial hepatectomy by gel mobility shift assay revealed a substantial reduction (more than 75% within 3 h) in the binding of the repressor to its cognate binding site. Our results suggest that a cis-acting transcriptional repressor in the promoter region of the mouse HGF gene is involved in cell-type-specific regulation through binding to its cognate trans-acting protein which exists in epithelial cells but is absent in fibroblast cells.

scholarly journals Structure, clustering and functional insights of repeats configurations in the upstream promoter region of the human coding genes

BMC Genomics ◽  
2018 ◽  
Vol 19 (S8) ◽  
Author(s):  
Fabian Tobar-Tosse ◽  
Patricia E. Veléz ◽  
Eliana Ocampo-Toro ◽  
Pedro A. Moreno
Keyword(s):  
Promoter Region ◽  
Upstream Promoter ◽  
Upstream Promoter Region

scholarly journals PhoP-Responsive Expression of the Salmonella enterica Serovar Typhimurium slyA Gene

2003 ◽  
Vol 185 (12) ◽  
pp. 3508-3514 ◽  
Author(s):  
Valia A. Norte ◽  
Melanie R. Stapleton ◽  
Jeffrey Green
Keyword(s):  
Transcription Factors ◽  
Salmonella Enterica ◽  
Promoter Region ◽  
External Signal ◽  
Dependent Manner ◽  
Factors Analysis ◽  
Upstream Promoter ◽  
Serovar Typhimurium ◽  
Transcription Regulators ◽  
Upstream Promoter Region

ABSTRACT The SlyA protein of Salmonella enterica serovar Typhimurium is a member of the MarR family of transcription regulators and is required for virulence and survival in professional macrophages. Isolated SlyA protein was able to bind a specific DNA target without posttranslational modification. This suggested that SlyA might not be activated by directly sensing an external signal but rather that the intracellular concentration of SlyA is enhanced in appropriate environments through the action of other transcription factors. Analysis of slyA transcription reveals the presence of a promoter region located upstream of the previously recognized SlyA repressed promoter. The newly identified upstream promoter region did not respond to SlyA but was activated by Mg(II) starvation in a PhoP-dependent manner. We present here evidence for a direct link between two transcription factors (PhoP and SlyA) crucial for Salmonella virulence.

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